Pleural Fluid Analysis in Chronic Hemothorax: A Mimicker of Infection.

OBJECTIVES: Timing to video-assisted thoracoscopic surgery (VATS) in hemothorax is based on preventing acute and long-term complications of retained blood products in the pleural space, including pleural space infection. We propose that the persistence of blood in the pleural space induces a proinflammatory state, independent of active infection. METHODS: We identified six patients with a hemothorax by clinical history, radiographic imaging, and pleural fluid analysis from a database of 1133 patients undergoing thoracentesis from 2002 to 2010 at the Medical University of South Carolina. RESULTS: In four of the six patients identified, the time from injury to thoracentesis was one, four, four, and five days, respectively. The fluid pH range was 7.32-7.41. The lactate dehydrogenase (LDH) range was 210-884 IU/L (mean 547 IU/L), and the absolute neutrophil count (ANC) range was 1196-3631 cells/µL. In two patients, the time from injury to thoracentesis was 7 and 60 days. In these two patients, the pH was 7.18 and 6.91, LDH was 1679 and 961 IU/L, and the ANC was 8134 and 5943 cells/µL. Microbiology and pathology were negative in all patients. CONCLUSIONS: The persistence of blood outside the vascular compartment, and within the pleural space, biochemically mirrors infection. We will explore the multiple mechanisms that account for development of pleural fluid acidosis, inflammation, and neutrophil recruitment.

Improving the predictive accuracy of identifying exudative effusions.

BACKGROUND: Application of Light's criteria results in misclassification of some transudative effusions as exudative, particularly because of congestive heart failure (CHF). We sought to determine if the serum to pleural fluid albumin (SF-A) and serum to pleural fluid protein (SF-P) gradients increased the predictive accuracy to correctly identify exudative effusions. METHODS: We retrospectively analyzed 1,153 consecutive patients who underwent a diagnostic thoracentesis at the Medical University South Carolina. Univariable logistic regression analyses were used to determine the statistical significance of pleural fluid tests that correctly identified exudative effusions. Tests with significant diagnostic accuracy were combined in multivariable logistic regression models, with calculation of areas under the curve (AUCs) to determine their predictive accuracy. The predictive capability of the best model was compared with Light's criteria and other test combinations. RESULTS: Pleural fluid lactate dehydrogenase (LDH), SF-A gradient, and SF-P gradient had a significant effect on the probability of identifying exudative pleural effusions. When combined together in a multivariable logistic regression, LDH (OR, 14.09 [95% CI, 2.25-85.50]), SF-A gradient (OR, 7.16 [95% CI, 1.24-41.43]), and SF-P gradient (OR, 6.83 [95% CI, 1.56-27.88]) had an AUC of 0.92 (95% CI, 0.85-0.98). CONCLUSIONS: Application of Light's criteria, not uncommonly, misclassifies CHF transudative effusions as exudates. In cases where no cause for an exudative effusion can be identified or CHF is suspected, the sequential application of the fluid LDH, followed by the SF-P and then the SF-A gradients, may assist in reclassifying pleural effusions as transudates.

Peripheral hyperpolarization-activated cyclic nucleotide-gated channels contribute to inflammation-induced hypersensitivity of the rat temporomandibular joint.

BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels conduct an inward cation current (Ih ) that contributes to the maintenance of neuronal membrane potential and have been implicated in a number of animal models of neuropathic and inflammatory pain. In the current study, we investigated HCN channel involvement in inflammatory pain of the temporomandibular joint (TMJ). METHODS: The contribution of HCN channels to inflammation (complete Freund's adjuvant; CFA)-induced mechanical hypersensitivity of the rat TMJ was tested with injections of the HCN channel blocker ZD7288. Retrograde labelling and immunohistochemistry was used to explore HCN channel expression in sensory neurons that innervate the TMJ. RESULTS: Injection of CFA into the TMJ (n = 7) resulted in a significantly increased mechanical sensitivity relative to vehicle injection (n = 7) (p < 0.05). The mechanical hypersensitivity generated by CFA injection was blocked by co-injection of ZD7288 with the CFA (n = 7). Retrograde labelling and immunohistochemistry experiments revealed expression predominantly of HCN1 and HCN2 channel subunits in trigeminal ganglion neurons that innervate the TMJ (n = 3). No change in the proportion or intensity of HCN channel expression was found in inflamed (n = 6) versus control (n = 5) animals at the time point tested. CONCLUSIONS: Our findings suggest a role for peripheral HCN channels in inflammation-induced pain of the TMJ. Peripheral application of a HCN channel blocker could provide therapeutic benefit for inflammatory TMJ pain and avoid side effects associated with activation of HCN channels in the central nervous system.

Peripheral N-methyl-d-aspartate receptors contribute to mechanical hypersensitivity in a rat model of inflammatory temporomandibular joint pain.

The aim of this study was to determine whether peripheral N-methyl-d-aspartate (NMDA) receptors are involved in inflammation-induced mechanical hypersensitivity of the temporomandibular joint (TMJ) region. We developed a rat model of mechanical sensitivity to Complete Freund's Adjuvant (CFA; 2µl containing 1µg Mycobacterium tuberculosis)-induced inflammation of the TMJ and examined changes in sensitivity following injection of NMDA receptor antagonists (dl-2-amino-5-phosphonovaleric acid (AP5) or Ifenprodil) with CFA. CFA injected into the TMJ resulted in an increase in mechanical sensitivity relative to pre-injection that peaked at day 1 and lasted for up to 3days (n=8, P<0.05). There was no change in mechanical sensitivity in vehicle-injected rats at any time-point (n=9). At day 1, there was a significant increase in mechanical sensitivity in animals injected with CFA+vehicle (n=7) relative to those injected with vehicle alone (n=7, P<0.05), and co-injection of AP5 (n=6) or Ifenprodil (n=7) with CFA blocked this hypersensitivity. Subcutaneous injection of AP5 (n=7) and Ifenprodil (n=5) instead of into the TMJ had no significant effect on CFA-induced hypersensitivity of the TMJ region. Western blot analysis revealed constitutive expression of the NR1 and NR2B subunits in trigeminal ganglion lysates. Immunohistochemical studies showed that 99% and 28% of trigeminal ganglion neurons that innervated the TMJ contained the NR1 and NR2B subunits respectively. Our findings suggest a role for peripheral NMDA receptors in inflammation-induced pain of the TMJ region. Targeting peripheral NMDA receptors with peripheral application of NMDA receptor antagonists could provide therapeutic benefit and avoid side effects associated with blockade of NMDA receptors in the central nervous system.

Stability of coronene at high temperature and pressure.

The infrared response of coronene (C(24)H(12)) under pressure and temperature conditions up to 10 GPa and 300 °C is examined in situ using a diamond anvil cell and synchrotron-source Fourier transform infrared (FTIR) spectroscopy. Coronene is a polycyclic aromatic hydrocarbon that is present in the interstellar medium and meteorites which may have contributed to the Earth's primordial carbon budget. It appears to undergo a reversible phase transition between 2 and 3.2 GPa at ambient temperature; new intramolecular bonds in the region 840-880 cm(-1) result from compression. We document the shift of spectral features to higher wavenumbers with increasing pressure but find this change suppressed by increased temperature. By investigating the stability of coronene over a range of naturally occurring conditions found in a range of environments, we assess the survival of the molecule through various terrestrial and extraterrestrial processes. Coronene has previously been shown to survive atmospheric entry during Earth accretion; this can now be extended to include survival through geological processes such as subduction and silicate melting of the rock cycle, opening the possibility of extraterrestrial coronene predating terrestrial accretion existing on Earth.

Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury-induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freund's adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased; compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.

A novel and in situ technique for the quantitative detection of MTBE and benzene degrading bacteria in contaminated matrices.

A novel and in situ technique is presented here as a better alternative to culture-dependent and PCR-based techniques for the quantitative detection of predominant bacterial species involved in the bioremediation of contaminants. It allowed rapid, specific and in situ identification of Biosep-immobilized eubacteria from MTBE- and benzene-contaminated matrices.

Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons.

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and ATP-mediated currents in sensory neurons have been shown to be mediated by both P2X3 and P2X2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X2 and P2X3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalization with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, non-peptidergic fibers (Bandeiraea simplicifolia isolectin B4; IB4). Forty percent of P2X2 and 64% of P2X3 receptor-expressing cells were IB4 positive, and 33% of P2X2 and 31% of P2X3 receptor-expressing cells were NF200 positive. Approximately 40% of cells expressing P2X2 receptors also expressed P2X3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labeled and 52% of these neurons expressed either P2X2 or P2X3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems.

Effects of sumatriptan on rat medullary dorsal horn neurons.

This study examined the cellular actions of the anti-migraine drug sumatriptan, on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis. Sumatriptan inhibited the miniature EPSC (mEPSC) rate in a dose dependent fashion, with an EC(50) of 250 nM. Sumatriptan (3 microM) inhibited the mEPSC rate by 36%, without altering the mEPSC amplitude. This effect was partially reversed by the 5HT(1D) specific antagonist BRL15572 (10 microM). In contrast, the 5HT(1B) agonist CP93129 (10 microm) did not alter the mEPSC rate. Furthermore, sumatriptan (3 microM) decreased the amplitude of electrically evoked EPSCs (eEPSC) by 40%. After incubating the slices in ketanserin (an antagonist which shows selectivity for 5HT(1D) over 5HT(1B) receptors) sumatriptan had little effect on eEPSC amplitude. In control conditions paired stimuli resulted in paired pulse depression (PPD; the ratio eEPSC(2)/eEPSC(1)=0.7+/-0.01), whilst in the presence of sumatriptan the PPD was blocked (ratio eEPSC(2)/eEPSC(1)=0.9+/-0.1). Sumatriptan produced no post-synaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (-60 to -130 mV). RT-PCR experiments revealed the presence of mRNA for both 5HT(1D) and 5HT(1B) receptor subtypes in the trigeminal ganglia and subnucleus caudalis. These data suggest that sumatriptan acts pre-synaptically on trigeminal primary afferent central terminals to reduce the probability of release of glutamate, and that this action is mediated through 5HT(1D) receptors.

Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3.

Vitamin D metabolites 1alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) regulate endochondral ossification in a cell maturation-dependent manner via membrane-mediated mechanisms. 24R,25(OH)(2)D(3) stimulates PKC activity in chondrocytes from the growth plate resting zone, whereas 1alpha,25(OH)(2)D(3) stimulates PKC in growth zone chondrocytes. We used the rat costochondral growth plate cartilage cell model to study how these responses are differentially regulated. 1alpha,25(OH)(2)D(3) acts on PKC, MAP kinase, and downstream physiological responses via phosphatidylinositol-specific PLC-beta; 24R,25(OH)(2)D(3) acts via PLD. In both cases, diacylglycerol (DAG) is increased, activating PKC. Both cell types possess membrane and nuclear receptors for 1alpha,25(OH)(2)D(3), but the mechanisms that render the 1alpha,25(OH)(2)D(3) pathway silent in resting zone cells or the 24R,25(OH)(2)D(3) pathway silent in growth zone cells are unclear. PLA(2) is pivotal in this process. 1alpha,25(OH)(2)D(3) stimulates PLA(2) activity in growth zone cells and 24R,25(OH)(2)D(3) inhibits PLA(2) activity in resting zone cells. Both processes result in PKC activation. To understand how negative regulation of PLA(2) results in increased PKC activity in resting zone cells, we used PLA(2) activating peptide to stimulate PLA(2) activity and examined cell response. PLAP is not expressed in resting zone cells in vivo, supporting the hypothesis that PLA(2) activation is inhibitory to 24R,25(OH)(2)D(3) action in these cells.

The actions of anandamide on rat superficial medullary dorsal horn neurons in vitro.

Whole-cell patch-clamp recordings were made from neurons in the trigeminal nucleus caudalis and trigeminal ganglion, in vitro, to investigate the cellular actions of the endogenous cannabinoid, anandamide. Anandamide has been shown to act through both the cannabinoid receptor 1 (CB1) and the vanilloid receptor 1 (VR1). Anandamide (30 microM) caused a 54 % increase in the rate of miniature excitatory post-synaptic currents (mEPSCs), without affecting their amplitude. The effect of anandamide was blocked by the VR1 antagonist capsazepine (20 microM), but not by the CB1-specific antagonist AM251 (3 microM). Application of the VR1 receptor agonist capsaicin (300 nM) caused a 4200 % increase in the mEPSC rate. In dissociated trigeminal ganglion neurons, both anandamide and capsaicin caused an outward current in neurons that were voltage clamped at +40 mV. The maximal outward current produced by anandamide (EC50, 10 microM) was 45 % of that produced by capsaicin (10 microM). Co-application of the VR1 antagonist capsazepine (30 microM) completely reversed the effects of both capsaicin and anandamide. The anandamide transport inhibitor, AM404 (30 microM) caused a 40 % increase in mEPSC rate in the slice preparation and an outward current in dissociated neurons. The latter current was reversed by the VR1 antagonist iodoresiniferatoxin (1 microM). The fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (20 microM) and OL53 (1 microM) did not enhance the effect of anandamide in either the slice or dissociated neuron preparations. These results suggest that within the superficial medullary dorsal horn, anandamide (30 microM) acts presynaptically to enhance the release of glutamate via activation of the VR1 receptor.

Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins.

DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes. Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication. Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome. Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function. A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats). These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

Cannabinoid actions on rat superficial medullary dorsal horn neurons in vitro.

1. This study examined the cellular actions of cannabinoids on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis, using whole-cell and perforated patch recording in brain slices. 2. The cannabinoid agonist WIN55,212-2 (3 microM) decreased the amplitude of both GABAergic and glycinergic electrically evoked inhibitory postsynaptic currents (IPSCs) by 35 and 41 %, respectively. This inhibition was completely reversed by the CB(1) receptor-selective antagonist N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) (SR141716A, 3 microM). WIN55,212-2 also produced relative facilitation of the second evoked IPSC to paired stimuli. 3. WIN55,212-2 decreased the rate of both GABAergic and glycinergic miniature IPSCs by 44 and 34 %, respectively, without changing their amplitude distributions or kinetics. 4. WIN55,212-2 did not affect the amplitude of electrically evoked non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). 5. WIN55,212-2 produced no postsynaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (-60 to -130 mV). 6. These results suggest that, within the superficial medullary dorsal horn, cannabinoids presynaptically inhibit GABAergic and glycinergic neurotransmission. At the cellular level, the analgesic action of cannabinoids on these medullary dorsal horn neurons therefore differs from that of mu-opioids, which have both pre- and postsynaptic actions.

Actions of nociceptin/orphanin FQ and other prepronociceptin products on rat rostral ventromedial medulla neurons in vitro.

1. Whole-cell patch clamp recordings were made from rat rostral ventromedial medulla (RVM) neurons in vitro to investigate the cellular actions of the opioid-like receptor ORL1 (NOP), ligand nociceptin/orphanin FQ and other putative prepronociceptin products. 2. Primary and secondary RVM neurons were identified as responding to the kappa-opioid receptor agonist U-69593 (300 nM to 1 microM) and the mu- and delta-opioid receptor agonist met-enkephalin (10 microM), respectively. Both primary and secondary RVM neurons responded to nociceptin (3 nM to 1 microM) with an outward current that reversed polarity at -115 mV in brain slices and with inhibition of Ca(2+) channel currents in acutely isolated cells. 3. The putative ORL1 antagonist J-113397 (1 microM) produced no change in membrane current and abolished the outward current produced by nociceptin (100 nM). In contrast, Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin-(1-13)NH(2) (300 nM to 1 microM) alone produced an outward current and partially reduced the outward current produced by nociceptin (300 nM) when co-applied. 4. In brain slices nociceptin (300 nM) reduced the amplitude of evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) but not non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs). 5. Met-enkephalin (10 microM), but not nociceptin (300 nM), reduced the rate of spontaneous miniature IPSCs in normal external potassium solution (K(+) 2.5 mM). In high external potassium (K(+) 17.5 mM), nociceptin reduced the rate of miniature IPSCs in the presence (Ca(2+) 2.4 mM, Mg(2+) 1.2 mM) but not in the absence of external calcium (Ca(2+) 0 mM, Mg(2+) 10 mM, Cd(2+) 10 microM). Nociceptin and met-enkephalin had no effect on the amplitude of miniature IPSCs. 6. The putative nociceptin precursor products nocistatin (rat prepronociceptin(125-132)) and rat prepronociceptin(154-181) had no effect on membrane currents, evoked IPSCs and evoked EPSCs. 7. These results indicate that nociceptin acts via the ORL1 receptor to directly inhibit both primary and secondary RVM neurons by activating a potassium conductance and by inhibiting calcium conductances. In addition, nociceptin inhibits GABA release within the RVM via a presynaptic Ca(2+)-dependent mechanism. Thus, nociceptin has the potential to exert both disinhibitory and inhibitory effects on neuronal action potential firing within the RVM.

Naso-ethmoid schwannoma with intracranial extension: case report.

Intranasal schwannomas are rare lesions, specially when they present with an intracranial extension. The fifth case in the medical literature of a naso-ethmoid schwannoma with extension into the anterior cranial fossa is presented. The magnetic resonance findings and the details of the combined intracranial / transfacial operative approach used are described. The possible origin and the clinical characteristics of this rare lesion are reviewed.

Postsynaptic K+ current induced by nociceptin in medullary dorsal horn neurons.

The actions of the endogenous ORL1 receptor (opioid receptor-like1) ligand nociceptin on the membrane properties of rat trigeminal nucleus caudalis neurons were examined by use of whole cell and perforated patch clamp recording in brain slices. Nociceptin produced an outward current in all neurons tested (EC50 112 nM). The outward current produced by nociceptin was completely reversed with the addition of the non-peptide ORL1 antagonist J-113397. Outward currents reversed polarity at -99+/-2 mV, close to the potential for K+ of -102 mV, suggesting that they were mediated by an increased K+ conductance. These results suggest that the analgesic action of nociceptin might be mediated by direct postsynaptic inhibition within the dorsal horn.

Remodeling of yeast genome expression in response to environmental changes.

We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition. These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves approximately 10% of yeast genes. The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change. These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.

Genome-wide location and function of DNA binding proteins.

Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.